Axmi-031, axmi-039, axmi-040 and axmi-049, a family of novel delta-endotoxin genes and methods for their use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a delta-endotoxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated delta-endotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/364,335, filed Feb. 2, 2009, which is a divisional of U.S.application Ser. No. 11/762,886, filed Jun. 14, 2007, now U.S. Pat. No.7,923,602, which claims the benefit of U.S. Provisional Application Ser.No. 60/813,774, filed Jun. 14, 2006, the contents of which are hereinincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA031US02N_SequenceListing.txt”, created on Feb. 14, 2012, and havinga size of 347 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Dipteran, and Coleopteran larvae. These proteins also have shownactivity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, andAcari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Höfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Because of the devastation that insects can confer there is a continualneed to discover new forms of Bacillus thuringiensis delta-endotoxins.

SUMMARY OF INVENTION

Compositions and methods for conferring pest resistance to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for delta-endotoxinpolypeptides, vectors comprising those nucleic acid molecules, and hostcells comprising the vectors. Compositions also include the polypeptidesequences of the endotoxin, and antibodies to those polypeptides. Thenucleotide sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. The nucleotide or amino acid sequences may besynthetic sequences that have been designed for expression in anorganism including, but not limited to, a microorganism or a plant.Compositions also comprise transformed bacteria, plants, plant cells,tissues, and seeds.

In particular, isolated nucleic acid molecules corresponding todelta-endotoxin nucleic acid sequences are provided. Additionally, aminoacid sequences corresponding to the polynucleotides are encompassed. Inparticular, the present invention provides for an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, or 38, a nucleotide sequence set forth in SEQ IDNO:1, 3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 37,or the delta-endotoxin nucleotide sequences deposited in bacterial hostsas Accession Nos. B-30935, B-30936, B-30937, and B-50046, as well asvariants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention are also encompassed.

Methods are provided for producing the polypeptides of the invention,and for using those polypeptides for controlling or killing alepidopteran or coleopteran pest. Methods and kits for detecting thenucleic acids and polypeptides of the invention in a sample are alsoincluded.

Methods for controlling or killing a nematode pest population arefurther provided. The methods comprise introducing into a plant apolynucleotide encoding a nematode-active polypeptide with a molecularsize greater than 22 kDa. These nematode-active polypeptides are usefulfor controlling or killing plant-parasitic nematodes, particularly cystnematodes.

The compositions and methods of the invention are useful for theproduction of organisms with pesticide resistance, specifically bacteriaand plants. These organisms and compositions derived from them aredesirable for agricultural purposes. The compositions of the inventionare also useful for generating altered or improved delta-endotoxinproteins that have pesticidal activity, or for detecting the presence ofdelta-endotoxin proteins or nucleic acids in products or organisms.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance in organisms, particularly plants or plantcells. The methods involve transforming organisms with a nucleotidesequence encoding a delta-endotoxin protein of the invention. Inparticular, the nucleotide sequences of the invention are useful forpreparing plants and microorganisms that possess pesticidal activity.Thus, transformed bacteria, plants, plant cells, plant tissues and seedsare provided. Compositions are delta-endotoxin nucleic acids andproteins of Bacillus thuringiensis. The sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of otherdelta-endotoxin genes, and for the generation of altered pesticidalproteins by methods known in the art, such as domain swapping or DNAshuffling. The proteins find use in controlling or killing lepidopteran,coleopteran, and nematode pest populations, and for producingcompositions with pesticidal activity.

Plasmids containing the nucleotide sequences of the invention weredeposited in the permanent collection of the Agricultural ResearchService Culture Collection, Northern Regional Research Laboratory(NRRL), 1815 North University Street, Peoria, Ill. 61604, United Statesof America, on Jun. 9, 2006, and assigned Accession Nos. NRRL B-30935(for axmi-031), NRRL B-30936 (for axmi-039), and NRRL B-30937 (foraxmi-040); and on May 29, 2007 and assigned NRRL B-50046 (axmi-049).These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112.

By “delta-endotoxin” is intended a toxin from Bacillus thuringiensisthat has toxic activity against one or more pests, including, but notlimited to, members of the Lepidoptera, Diptera, and Coleoptera ordersor members of the Nematoda phylum, or a protein that has homology tosuch a protein. In some cases, delta-endotoxin proteins have beenisolated from other organisms, including Clostridium bifermentans andPaenibacillus popilliae. Delta-endotoxin proteins include amino acidsequences deduced from the full-length nucleotide sequences disclosedherein, and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start site,or due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin, or in the pest after ingestion of the protein. Delta-endotoxinsinclude proteins identified as cry1 through cry43, cyt1 and cyt2, andCyt-like toxin. There are currently over 250 known species ofdelta-endotoxins with a wide range of specificities and toxicities. Foran expansive list see Crickmore et al. (1998), Microbiol. Mol. Biol.Rev. 62:807-813, and for regular updates see Crickmore et al. (2003)“Bacillus thuringiensis toxin nomenclature,” atwww.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

Provided herein are novel isolated nucleotide sequences that conferpesticidal activity. Also provided are the amino acid sequences of thedelta-endotoxin proteins. The protein resulting from translation of thisgene allows cells to control or kill pests that ingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding delta-endotoxinproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify delta-endotoxin encoding nucleic acids. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolateddelta-endotoxin encoding nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A delta-endotoxinprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of non-delta-endotoxin protein (also referred to herein as a“contaminating protein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1, 3, 5, 34, and 37, thedelta endotoxin nucleotide sequences deposited in bacterial hosts asAccession Nos. NRRL B-30935, B-30936, B-30937, and B-50046, andvariants, fragments, and complements thereof (for example, SEQ ID NO:7,9, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32). By “complement” isintended a nucleotide sequence that is sufficiently complementary to agiven nucleotide sequence such that it can hybridize to the givennucleotide sequence to thereby form a stable duplex. The correspondingamino acid sequence for the delta-endotoxin protein encoded by thisnucleotide sequence are set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 37.

Nucleic acid molecules that are fragments of these delta-endotoxinencoding nucleotide sequences are also encompassed by the presentinvention (for example, SEQ ID NO:9, 11, 14, 18, 20, 22, and 24). By“fragment” is intended a portion of the nucleotide sequence encoding adelta-endotoxin protein. A fragment of a nucleotide sequence may encodea biologically active portion of a delta-endotoxin protein, or it may bea fragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. Nucleic acid molecules that are fragments of adelta-endotoxin nucleotide sequence comprise at least about 50, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800,1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400,2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000,3050, 3100, 3150, 3200, 3250, 3300, 3350 contiguous nucleotides, or upto the number of nucleotides present in a full-length delta-endotoxinencoding nucleotide sequence disclosed herein (for example, 3558nucleotides for SEQ ID NO:1, 3984 nucleotides for SEQ ID NO:3, 3720nucleotides for SEQ ID NO:5, and 3669 nucleotides for SEQ ID NO:34)depending upon the intended use. By “contiguous” nucleotides is intendednucleotide residues that are immediately adjacent to one another.Fragments of the nucleotide sequences of the present invention willencode protein fragments that retain the biological activity of thedelta-endotoxin protein and, hence, retain pesticidal activity. By“retains activity” is intended that the fragment will have at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the pesticidal activity of the delta-endotoxin protein.Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J.of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

A fragment of a delta-endotoxin encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100 contiguous amino acids, or up to the total number ofamino acids present in a full-length delta-endotoxin protein of theinvention (for example, 1185 amino acids for SEQ ID NO:2, 1327 aminoacids for SEQ ID NO:4, 1239 amino acids for SEQ ID NO:6, and 1223 aminoacids for SEQ ID NO:35).

Preferred delta-endotoxin proteins of the present invention are encodedby a nucleotide sequence sufficiently identical to the nucleotidesequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, or 37. By “sufficiently identical” is intended an amino acidor nucleotide sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous todelta-endotoxin-like nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous todelta-endotoxin protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389. Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. SeeAltschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., BLASTX and BLASTN) can be used. Alignment may also be performedmanually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10. The invention alsoencompasses variant nucleic acid molecules (for example, SEQ ID NO:7, 9,11, 16, 18, 22, 24, 26, 28, 30, and 32). “Variants” of thedelta-endotoxin encoding nucleotide sequences include those sequencesthat encode the delta-endotoxin proteins disclosed herein but thatdiffer conservatively because of the degeneracy of the genetic code aswell as those that are sufficiently identical as discussed above.Naturally occurring allelic variants can be identified with the use ofwell-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the delta-endotoxin proteinsdisclosed in the present invention as discussed below. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, retaining pesticidal activity. By “retains activity”is intended that the variant will have at least about 30%, at leastabout 50%, at least about 70%, or at least about 80% of the pesticidalactivity of the native protein. Methods for measuring pesticidalactivity are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem.J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology78:290-293; and U.S. Pat. No. 5,743,477, all of which are hereinincorporated by reference in their entirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodeddelta-endotoxin proteins, without altering the biological activity ofthe proteins. Thus, variant isolated nucleic acid molecules can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of a delta-endotoxin protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of the amino acidsequences of the present invention and known delta-endotoxin sequences.Examples of residues that are conserved but that may allow conservativeamino acid substitutions and still retain activity include, for example,residues that have only conservative substitutions between all proteinscontained in an alignment of the amino acid sequences of the presentinvention and known delta-endotoxin sequences. However, one of skill inthe art would understand that functional variants may have minorconserved or nonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer delta-endotoxin activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingdelta-endotoxin sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the delta-endotoxin nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known delta-endotoxin-encodingnucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in the nucleotidesequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,or 400 consecutive nucleotides of delta-endotoxin encoding nucleotidesequence of the invention or a fragment or variant thereof. Methods forthe preparation of probes for hybridization are generally known in theart and are disclosed in Sambrook and Russell, 2001, supra hereinincorporated by reference.

For example, an entire delta-endotoxin sequence disclosed herein, or oneor more portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding delta-endotoxin-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, or at least about 20nucleotides in length. Such probes may be used to amplify correspondingdelta-endotoxin sequences from a chosen organism by PCR. This techniquemay be used to isolate additional coding sequences from a desiredorganism or as a diagnostic assay to determine the presence of codingsequences in an organism. Hybridization techniques include hybridizationscreening of plated DNA libraries (either plaques or colonies; see, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Delta-endotoxin proteins are also encompassed within the presentinvention. By “delta-endotoxin protein” is intended a protein having theamino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, or 38. Fragments, biologicallyactive portions, and variants thereof are also provided, and may be usedto practice the methods of the present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 16, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, or 38 and that exhibit pesticidalactivity (for example, SEQ ID NO:15, 19, 21, 23, or 25). A biologicallyactive portion of a delta-endotoxin protein can be a polypeptide thatis, for example, 10, 25, 50, 100 or more amino acids in length. Suchbiologically active portions can be prepared by recombinant techniquesand evaluated for pesticidal activity. Methods for measuring pesticidalactivity are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety. As used here, a fragment comprises at least8 contiguous amino acids of SEQ ID NO:2, 4, 6, 8, 10, 12, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, or 38. The invention encompasses otherfragments, however, such as any fragment in the protein greater thanabout 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, or 1300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 16, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, or 38 (e.g., SEQ ID NO:10, 12, 17, 19,23, 25, 27, 29, 31, or 33). Variants also include polypeptides encodedby a nucleic acid molecule that hybridizes to the nucleic acid moleculeof SEQ ID NO:1, 3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, or 37, or a complement thereof, under stringent conditions. Variantsinclude polypeptides that differ in amino acid sequence due tomutagenesis. Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, retaining pesticidalactivity. Methods for measuring pesticidal activity are well known inthe art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

Bacterial genes, such as the axmi-031, axmi-039, axmi-040, and axmi-049genes of this invention, quite often possess multiple methionineinitiation codons in proximity to the start of the open reading frame.Often, translation initiation at one or more of these start codons willlead to generation of a functional protein. These start codons caninclude ATG codons. However, bacteria such as Bacillus sp. alsorecognize the codon GTG as a start codon, and proteins that initiatetranslation at GTG codons contain a methionine at the first amino acid.Furthermore, it is not often determined a priori which of these codonsare used naturally in the bacterium. Thus, it is understood that use ofone of the alternate methionine codons may also lead to generation ofdelta-endotoxin proteins that encode pesticidal activity. Thesedelta-endotoxin proteins are encompassed in the present invention andmay be used in the methods of the present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a delta-endotoxin may be alteredby various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a delta-endotoxin of the present invention. This proteinmay be altered in various ways including amino acid substitutions,deletions, truncations, and insertions of one or more amino acids of SEQID NO:2, 4, 6, 8, 10, 12, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or38, including up to about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 100, about 105,about 110, about 115, about 120, about 125, about 130 or more amino acidsubstitutions, deletions or insertions.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a delta-endotoxin protein canbe prepared by mutations in the DNA. This may also be accomplished byone of several forms of mutagenesis and/or in directed evolution. Insome aspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of a delta-endotoxin to confer pesticidal activity may beimproved by the use of such techniques upon the compositions of thisinvention. For example, one may express a delta-endotoxin in host cellsthat exhibit high rates of base misincorporation during DNA replication,such as XL-1 Red (Stratagene). After propagation in such strains, onecan isolate the delta-endotoxin DNA (for example by preparing plasmidDNA, or by amplifying by PCR and cloning the resulting PCR fragment intoa vector), culture the delta-endotoxin mutations in a non-mutagenicstrain, and identify mutated delta-endotoxin genes with pesticidalactivity, for example by performing an assay to test for pesticidalactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests. Examples of mutations that result in increasedtoxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev.62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent delta-endotoxin protein coding regions can be used to create anew delta-endotoxin protein possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled between adelta-endotoxin gene of the invention and other known delta-endotoxingenes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased insecticidal activity.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating altereddelta-endotoxin proteins. Domains II and III may be swapped betweendelta-endotoxin proteins, resulting in hybrid or chimeric toxins withimproved pesticidal activity or target spectrum. Methods for generatingrecombinant proteins and testing them for pesticidal activity are wellknown in the art (see, for example, Naimov et al. (2001) Appl. Environ.Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ.Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem.266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930;Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925).

Vectors

A delta-endotoxin sequence of the invention may be provided in anexpression cassette for expression in a plant of interest. By “plantexpression cassette” is intended a DNA construct that is capable ofresulting in the expression of a protein from an open reading frame in aplant cell. Typically these contain a promoter and a coding sequence.Often, such constructs will also contain a 3′ untranslated region. Suchconstructs may contain a “signal sequence” or “leader sequence” (i.e.,SEQ ID NO:9, 11, 28, 30, and 32) to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.By “leader sequence” is intended any sequence that when translated,results in an amino acid sequence (i.e., SEQ ID NO:10, 12, 29, 31, and33) sufficient to trigger co-translational transport of the peptidechain to a sub-cellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the delta-endotoxin sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the delta-endotoxin is targeted to the chloroplastfor expression. In this manner, where the delta-endotoxin is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe delta-endotoxin to the chloroplasts. Such transit peptides are knownin the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol.Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550;Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al.(1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al.(1986) Science 233:478-481.

The delta-endotoxin gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The delta-endotoxin gene of the inventionmay be modified to obtain or enhance expression in plant cells.Typically a construct that expresses such a protein would contain apromoter to drive transcription of the gene, as well as a 3′untranslated region to allow transcription termination andpolyadenylation. The organization of such constructs is well known inthe art. In some instances, it may be useful to engineer the gene suchthat the resulting peptide is secreted, or otherwise targeted within theplant cell. For example, the gene can be engineered to contain a signalpeptide to facilitate transfer of the peptide to the endoplasmicreticulum. It may also be preferable to engineer the plant expressioncassette to contain an intron, such that mRNA processing of the intronis required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the delta-endotoxin are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the delta-endotoxin is then tested by hybridizing the filterto a radioactive probe derived from a delta-endotoxin, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thedelta-endotoxin gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thedelta-endotoxin protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a delta-endotoxin that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a delta-endotoxin may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a delta-endotoxin may be tested for pesticidalactivity, and the plants showing optimal activity selected for furtherbreeding. Methods are available in the art to assay for pest activity.Generally, the protein is mixed and used in feeding assays. See, forexample Marrone et al. (1985) J. of Economic Entomology 78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in Pest Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing adelta-endotoxin gene into a cellular host. Expression of thedelta-endotoxin gene results, directly or indirectly, in theintracellular production and maintenance of the pesticide. In one aspectof this invention, these cells are then treated under conditions thatprolong the activity of the toxin produced in the cell when the cell isapplied to the environment of target pest(s). The resulting productretains the toxicity of the toxin. These naturally encapsulatedpesticides may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target pest,e.g., soil, water, and foliage of plants. See, for example EPA 0192319,and the references cited therein. Alternatively, one may formulate thecells expressing a gene of this invention such as to allow applicationof the resulting material as a pesticide.

Nematode Control

Plant-parasitic nematodes, including cyst nematodes, root knotnematodes, and other nematodes, cause billions of dollars in damage tocrops every year. Plant-parasitic nematodes pierce plant cell walls withtheir stylet, which is formed by some of the mouth and esophagus parts,and pump up the plant cell just into their digestive system. Numerouspublications in the art conclude that sedentary plant parasiticnematodes do not ingest molecules larger than about 23-28 kDa in vivo.This has led to the widespread belief that the feeding tube produced bythese nematodes acts as a molecular sieve, restricting the size ofmolecules that can be ingested. Bockenhoff and Grundler ((1994)Parasitology 109:249-254) injected fluorescently labeled dextrans intosyncytia that were being fed upon by Heterodera schachtii. They foundthat the nematodes ingested 22 kDa dextrans, but not 40 kDa dextrans.Urwin et al. ((1998) Planta 204:472-479) found that a 23 kDa fusionprotein expressed in transgenic Arabidopsis could not be ingested by H.schachtii, although a protein of about 11 kDa could be ingested.Similarly, Unwin et al. ((1997) Molecular Plant-Microbe Interactions10:394-400) found that 28 kDa green fluorescent protein (GFP) expressedin transgenic Arabidopsis could not be ingested by H. schachtii. Thus,it was not previously recognized or demonstrated that nematodes could becontrolled by expressing a protein greater than 22 kDa in a plant sinceit was understood that nematodes would not ingest such a protein.

Provided herein are methods and compositions for conferring resistanceto nematodes in plants. The methods comprise introducing into a plant atleast one nucleotide sequence encoding a nematode-active polypeptide andgrowing the plant in a field containing nematodes. By “nematode-activepolypeptide” is intended a polypeptide that, when ingested by thenematode, results in the death or stunting of growth or proliferation ofat least one nematode. In one embodiment, the nematode-activepolypeptide has a molecular weight greater than about 22 kDa, about 23kDa, about 24, about 25, about 26, about 27, about 28, about 29, about30, about 31, about 32, about 33, about 34, about 35, about 36, about37, about 38, about 39, about 40, about 41, about 42, about 43, about44, about 45, about 46, about 47, about 48, about 49, about 50, about51, about 52, about 53, about 54, about 55, about 56, about 57, about58, about 59, about 60, about 61, about 62, about 63, about 64, about65, about 66, about 67, about 68, about 69, about 70, about 71, about72, about 73, about 74, about 75, about 76, about 77, about 78, about79, about 80 kDa, or greater.

In another embodiment, the nematode-active polypeptide has activityagainst plant-parasitic nematodes. Expression of the nematode-activepolypeptide in a plant reduces the ability of nematodes to infest orfeed on roots of the plant. Examples of plant-parasitic nematodessensitive to the compositions of the present invention include root knotnematodes (Meloidogyne sp.), stunt nematode (Tylenchorhynchus sp.),lance nematode (Hoplolaimus sp.), spiral nematode (Helicotylenchus sp.),lesion nematode (Pratylenchus sp.), cyst nematode (Heterodera sp. andGlobodera sp.), and ring nematode (Criconema sp.).

Pesticidal Compositions

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, coleopteran, or nematode pests may be killed or reduced innumbers in a given area by the methods of the invention, or may beprophylactically applied to an environmental area to prevent infestationby a susceptible pest. Preferably the pest ingests, or is contactedwith, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrinidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylinidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseintroducing into a plant or plant cell a polynucleotide comprising apesticidal sequence disclosed herein. As defined herein, the “yield” ofthe plant refers to the quality and/or quantity of biomass produced bythe plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a pesticidal protein disclosedherein. Expression of the pesticidal protein results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Growth of ATX9387, and Preparation of Extracts

Strain ATX9387, identified as a member of the Bacillus cereus /Bacillusthuringiensis group by MIDI analysis, was grown in T3 medium at 30degrees for times ranging from 16 hours to 5 days. Cultures werecentrifuged and the supernatants were passed through 0.2 micron filters,resulting in sterile supernatants

Example 2 C. elegans Bioassay

Caenorhabitis elegans (“C. elegans”) hermaphrodites were reared as knownin the art, to generate populations of healthy animals for bioassay.General procedures for growth, harvesting, and genetic manipulation ofC. elegans including growth media, etc. may be found in the art, forexample, in Wood, ed. (1988) The Nematode Caenorhabditis elegans (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Sterile supernatants from strain ATX9387 were tested for activity on C.elegans. Bioassays were performed in 96-well plates. Five to tennematodes were added to 80 μl of S medium (Wood, supra) and were mixedwith 20 μl of sterile supernatant, 0.5 μl of concentrated HB101(prepared as described in Wood, supra) and rifampicin (finalconcentration of 0.1 μg/μl). Assays were allowed to proceed at roomtemperature for 3 days and nematodes were quantitated. Negative controlsamples (T3 medium or sterile supernatants from inactive strains)contained hundreds of active nematodes, while test samples (containingATX9387 supernatant) contained 5 to 10 nematodes that were sluggish ordead. The results of the bioassay of ATX9387 extracts on C. elegans areshown in Table 1.

TABLE 1 Activity of ATX9387 extracts on C. elegans Growth Time ATX9387Control 16 hours − − 1 day − − 2 days − − 3 days ++ − 4 days ++ − 5 days++ −

Example 3 Activity of ATX9387 on Soybean Cyst Nematodes

A sterile supernatant of a 5-day culture of ATX9387 was concentrated40-fold and fed to SCN J2 nematodes. Nematodes feeding on sterilesupernatant were reproducibly observed to be sluggish, and exhibitedhigher motility than nematodes fed extract of a negative controlconcentrated to the same extent.

Example 4 Anti-Nematode Activity from ATX9387 is Conferred by a Protein

To identify the anti-nematode activity in ATX9387, the following testswere performed. First, the ability of the activity to be destroyed byheating was tested by heating samples of sterile supernatant fromATX9387 to 100° C. for 10 minutes, then assaying heated material in anematode bioassay. Heat treatment of sterile supernatants from ATX9387resulted in a loss of anti-nematode activity. Next, active samples weretreated with pronase to degrade proteins. This treatment resulted inloss of activity. Sterile supernatants from ATX9387 were passed througha 3 kDa molecular weight cut-off (“MWCO”) concentration unit. The activeingredient was retained by the 3 kDa MWCO filter while the flow-throughshowed no activity. This indicates that the active molecule was largerthan 3 kDa in size.

Example 5 Fractionation of Activity from ATX9387

The sterile supernatant of a 4-day culture of ATX9387 was fractionatedby liquid chromatography on an anion exchange column in 20 mM Tris pH 8,using a gradient from 0 M to 1 M NaCl. Several consecutive fractionswere active. The most active fraction was subjected to SDS-PAGE, and twoprominent bands of approximately 130 kd and approximately 70 kd wereobserved.

In another experiment strain ATX9387 was grown for 5 days in T3 mediumat 30° C. The culture was centrifuged at 8,000×g for 10 minutes, and thesupernatant was passed through a 0.2 μm filter. The filtered supernatantwas dialyzed against 20 mM Tris pH 8 using Spectra/Por 1 dialysis tubing(6-8,000 MWCO), and was then fractionated on an anion exchange column(Mono Q) using a gradient from 0 to 1 M NaCl over 20 bed volumes. Thefractions were dialyzed against 20 mM Tris pH 8 using Slide-A-Lyzer(Pierce Biotechnology, Rockford, Ill.) mini dialysis units (7,000 MWCO),and bioassayed on C. elegans using 20 μl of sample in a 100 μl bioassayvolume, as described herein. Fractions 12 and 13 were found to beactive, and were pooled and then concentrated 7-fold using an AmiconUltra-4 5,000 MWCO concentrator. The concentrated material wasfractionated on a gel filtration column (Superdex 200) in 50 mM sodiumphosphate, 150 mM NaCl, pH 7. Fractions were concentrated 10-fold usingCentricon YM-3 concentrators, and were bioassayed on C. elegans asabove. Fractions 5 and 6 were the most active, and fraction 7 wassomewhat active. SDS-PAGE of the fractions showed that fractions 5-7shared several proteins: a protein at about 130 kDa, a doublet at about75 kDa, and a protein at about 53 kDa. The most prominent bands weresubjected to N-terminal protein sequencing by Edman degradation. The 130kDa protein and the 70 kDa protein had very similar N-terminal sequences(cysteine could not be detected by the method used), and thus are likelyto result from the same initial protein. This protein was designated asAXMI-031 (SEQ ID NO:2).

TABLE 2 N-terminal sequence of the ~130 kDa protein from ATX9387 1 A,S + S′, M 2 D, Q 3 ?, P, N 4 N 5 L 6 Q 7 S 8 Q 9 ?, Q 10 N 11 I 12 P 13Y 14 N 15 V

TABLE 3 N-terminal sequence of the about ~70 kDa protein from ATX9387 1A, G, S + S′ 2 F 3 P 4 N 5 L 6 Q 7 V? 8 Q 9 ? 10 N?, V? 11 I 12 P 13 Y,Q 14 ?, N 15 ?, VA search of protein databases with the N-terminal sequences of the ˜130kDa protein and the ˜70 kDa protein demonstrated that the N-terminalamino acids of the AXMI-031 proteins have significant similarity to theN-terminus of the endotoxin Cry14Aa (SEQ ID NO:13).

N-terminal sequence of Cry14A: MDCNLQSQQNIPYNV (amino acid residues 1through 15 of SEQ ID NO:13).

Example 6 Cloning of the axmi-031 Coding Region from ATX9387

A random fragment library of ATX9387 was generated, and a DNA clone(pAX031) containing the DNA sequence encoding the N-terminus of axmi-031was identified. A second clone, pAX032, was identified as containing theC-terminus of axmi-031. Clones pAX031 and pAX032 overlap substantially,such that it is clear to one skilled in the art that both clonestogether comprise the entire axmi-031 coding region.

To confirm the nature of the AXMI-031, a genomic clone was amplifiedusing a high fidelity polymerase PFU ULTRA™ (Stratagene) from total DNAusing primers designed to the ends of the axmi-031 open reading frame.The resulting PCR product was cloned into the PCR-TOPOII-Blunt vector(Invitrogen) to create pAX980. The DNA sequence pAX980 was determinedand found to contain the same open reading frame as in the randomfragment library clones pAX031 and pAX032. Translation of this openreading frame generates a protein sequence consistent with theN-terminal sequence obtained from the purified AXMI-031 protein. Thus,this open reading frame was designated axmi-031 (SEQ ID NO:1). Theplasmid clone pAX2515, containing axmi-031 was deposited on Jun. 9, 2006and assigned the accession number NRRL B-30935.

Example 7 Comparison of AXMI-031 to Other Known Endotoxins

Database searches using the AXMI-031 protein sequence demonstrate thatAXMI-031 is a member of the delta-endotoxin class of insecticidalproteins. AXMI-031 is most similar to the cry14Aa1 endotoxin. The aminoacid sequence of AXMI-031 is 86.6% identical to CRY14Aa1 (SEQ ID NO:13).

Example 8 Expression of the AXMI-031 Polypeptide in E. coli

For soluble expression in E. coli, primers were designed to include thetranslation start and stop codons from the axmi-0310RF. The primersadded an optimal RBS and Gateway attB recombination sites. StratagenePfu I polymerase was used to consistently amplify the ORF from thepAX980, and recombined with pDONR221 (Invitrogen, Carlsbad, Calif.) tocreate the entry vector pAX2515 (as per protocols from Invitrogen). Theclone was sequenced for verification. A further recombination wasperformed to introduce the ORF into pDEST17 to be expressed by the T7promoter, yielding pAX2530. The presence and orientation of the insertedaxmi-031 fragment were verified by restriction digest, and transformedinto E. coli BL21 (DE3) cells. This vector produces a translationalfusion of 26 amino acids (3.17 kDa), including a 6×His tag, on theN-terminus of AXMI-031.

His-AXMI-031 was expressed from pAX2530 in BL21*(DE3) cells as follows.A starter culture of pAX2530 was grown overnight at 37° C. in LB with 50μg/ml carbenicillin. The following day the saturated culture was diluted1:100 into fresh medium, and the fresh culture grown at 37° to an OD of0.4, at which time the culture was placed at room temperature withshaking overnight. As a control, an expression vector carrying theaxmi-004 gene (pAX2530) was constructed as for axmi-031, and theAXMI-004 protein (U.S. patent application Ser. No. 10/782,020) wasexpressed from pAX2504 in the same E. coli host. Whole cultures wereanalyzed by SDS-PAGE. Cultures containing pAX2530 produced a prominentprotein band at approximately 135 kDa, the expected size forHis-AXMI-031 protein, while cultures containing pAX2504 did not.

Example 9 Antibodies to AXMI-031

To generate anti-AXMI-031 antibodies, affinity purified AXMI-031 (SEQ IDNO:2) protein was inoculated into rabbits, and antisera to AXMI-031isolated and titered as known in the art. The selected antisera werefound to also react with SYNAXMI-031 protein (SEQ ID NO:8).

Example 10 Activity of AXMI-031 on C. elegans

Cultures of E. coli containing pAX2530, were prepared, and found toproduce a 135 kDa protein not present in control strains, suggesting theexpression of AXMI-031 in pAX2530 containing strains. These cultureswere tested for activity on C. elegans and were active against C.elegans, while control cultures showed no activity against C. elegans.

Example 11 Expression of AXMI-031 in Bacillus

The insecticidal gene axmi-031 is amplified by PCR from pAX980, and thePCR product is cloned into the Bacillus expression vector pAX916, oranother suitable vector, by methods well known in the art. The resultingBacillus strain, containing the vector with axmi-031 is cultured on aconventional growth media, such as CYS media (10 g/l Bacto-casitone; 3g/l yeast extract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mMMnCl₂; 0.05 mM FeSO₄), until sporulation is evident by microscopicexamination. Samples are prepared, and AXMI-031 protein was tested foractivity in bioassays against C. elegans, and found to have activity.

Example 12 Synaxmi-031, Synaxmi-031(apo) and Synaxmi-031 (ER)

In one aspect of the invention, synthetic axmi-031 sequences aregenerated, for example synaxmi-031 (SEQ ID NO:7). These syntheticsequences have an altered DNA sequence relative to the axmi-031sequence, and encode a protein that is collinear with the originalAXMI-031 protein, but lacks the C-terminal “crystal domain” present inAXMI-031. The synaxmi-031 gene sequence encodes SYNAXMI-031 protein (SEQID NO:8), which comprises the first 685 amino acids of the AXMI-031protein.

In another aspect of the invention, modified versions of synaxmi-031 aredesigned such that the resulting peptide is targeted to a plantorganelle, such as the endoplasmic reticulum or the apoplast. Peptidesequences known to result in targeting of fusion proteins to plantorganelles are known in the art. For example, the N-terminal region ofthe acid phosphatase gene from the White Lupin Lupinus albus (GenebankID GI:14276838; Miller et al. (2001) Plant Physiology 127: 594-606) isknown in the art to result in endoplasmic reticulum targeting ofheterologous proteins. If the resulting fusion protein also contains anendoplasmic retention sequence comprising the peptideN-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the “KDEL”motif (SEQ ID NO:36) at the C-terminus, the fusion protein will betargeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Thus, the synaxmi-031ER gene (SEQ ID NO:11) encodes a fusion proteinthat contains the N-terminal thirty-one amino acids of the acidphosphatase gene from the White Lupin Lupinus albus fused to theN-terminus of SYNAXMI-031, as well as the KDEL sequence at theC-terminus. Thus, the resulting protein AXMI-031ER (SEQ ID NO:12), ispredicted to be targeted the plant endoplasmic reticulum upon expressionin a plant cell.

The synaxmi-031(apo) gene (SEQ ID NO:9) encodes a fusion protein thatcontains the N-terminal thirty-one amino acids of the acid phosphatasegene from the White Lupin Lupinus albus fused to the N-terminus ofSYNAXMI-031, but lacks the KDEL sequence at the C-terminus. Thus, theresulting protein AXMI-031(APO) (SEQ ID NO:10), is predicted to betargeted to the plant apoplast upon expression in a plant cell.

Example 13 Truncations of Synaxmi-031 to Yield Alternate AXMI-031Proteins

DNA constructs that resulted in expression of variants of AXMI-031protein were developed and expressed, in addition to synthetic sequencesencoding AXMI-031 and variants and fragments thereof (SEQ ID NO:15-27).A subset of these genes were tested for nematode activity in vitro.

TABLE 4 Nematicidal Activity of AXMI-031 variants in vitro NucleotideAmino acid SEQ ID SEQ ID Active on C. Protein NO: NO: elegans? BacterialExpression AXMI-031-truncated 14 15 Yes AXMI-031(m1) 16 17 YesAXMI-031(m1)- 18 19 Yes truncated AXMI-031(A-D) 20 21 YesSYNAXMI-031(A-D) 26 27 NT AXMI-031(B-C) 22 23 Yes AXMI-031(B-D) 24 25Yes NT = not tested

Example 14 Truncations and Addition of Cellular Targeting Domain(s) forPlant Expression

In another aspect of the invention, modified versions of the synaxmi-031sequences are designed such that the resulting peptide is targeted to aplant organelle, such as the endoplasmic reticulum or the apoplast.

In another aspect of the invention, the genes are truncated such thatthe resulting peptide is a truncated version of AXMI-031, which may ormay not be further modified for targeting to plant organelles, such asthe apoplast or the endoplasmic reticulum.

In another aspect of the invention, modified versions are developed thatresult in expression of truncated variants that contain domains designedto target the resulting protein to plant organelles.

The following variant nucleotide sequences were designed:

aposynaxmi-031(A-D) (SEQ ID NO:28) encodes the APOAXMI-031(A-D) protein(SEQ ID NO:29). apoSyn2axmi-031(A-D) (SEQ ID NO:30) encodes theAPOAXMI-031(A-D) protein (SEQ ID NO:31). aposynaxmi-031(fl) (SEQ IDNO:37) encodes the APOSYNAXMI-031(FL) protein (SEQ ID NO:38).Synaxmi031(fl)-ER (SEQ ID NO:32) encodes the SYNAXMI-031(FL)-ER protein(SEQ ID NO:33).

Example 15 Extraction of Plasmid DNA from Strains ATX16538, ATX16093 andATX21049

Strains ATX16538, ATX16093, and ATX21049 were selected for analysis.Pure cultures of each strain were grown in large quantities of richmedia. The cultures were centrifuged to harvest the cell pellet. Thecell pellet was then prepared by treatment with SDS by methods known inthe art, resulting in breakage of the cell wall and release of DNA.Proteins and large genomic DNA was then precipitated by a high saltconcentration. The plasmid DNA was then precipitated with ethanol. Inseveral instances, the plasmid DNA was separated from any remainingchromosomal DNA by high-speed centrifugation through a cesium chloridegradient. Alternatively, the plasmid DNA was purified by binding to aresin, as known in the art. For each strain, the quality of the DNA waschecked by visualization on an agarose gel by methods known in the art.

Example 16 Cloning of Genes from Strains ATX16538, ATX16093, andATX21049

DNA libraries were prepared from the plasmid DNA or each strain. Thismay be achieved in many ways as known in the art. For, example, thepurified plasmid DNA can be sheared into 5-10 kb sized fragments and the5′ and 3′ single stranded overhangs repaired using T4 DNA polymerase andKlenow fragment in the presence of all four dNTPs, as known in the art.Phosphates can then be attached to the 5′ ends by treatment with T4polynucleotide kinase, as known in the art. The repaired DNA fragmentscan then be ligated overnight into a standard high copy vector (i.e.pBLUESCRIPT® SK+), suitably prepared to accept the inserts as known inthe art (for example by digestion with a restriction enzyme producingblunt ends).

The quality of the resulting DNA libraries was analyzed by digesting asubset of clones with a restriction enzyme known to have a cleavage siteflanking the cloning site. A high percentage of clones were determinedto contain inserts, usually with an average insert size of 5-6 kb.

Example 17 High Throughput Sequencing of Library Plates

Once the DNA library quality was checked and confirmed, colonies weregrown in a rich broth in 2 ml 96-well blocks overnight at 37° C.,typically at a shaking speed of 350 rpm. The blocks were centrifuged tocollect the cells at the bottom of the block. The blocks were thenprepared by standard alkaline lysis prep in a high throughput format.

The end sequences of clones from this library were then determined for alarge number of clones from each block in the following manner: The DNAsequence of each clone chosen for analysis was determined using thefluorescent dye terminator sequencing technique (Applied Biosystems), bymethods known in the art using an automated DNA sequencing machine, andstandard oligonucleotide primers that anneal to the plasmid vector inthe region flanking the insert.

Example 18 Assembly and Screening of Sequencing Data

DNA sequences obtained were compiled into an assembly project andaligned together to form contigs. This can be done efficiently using acomputer program, such as Vector NTi, or alternatively by using thePred/Phrap suite of DNA alignment and analysis programs as describedelsewhere herein. These contigs, along with any individual read that maynot have been added to a contig, were compared to a compiled database ofall classes of known pesticidal genes. Contigs or individual readsidentified as having identity to a known endotoxin or pesticidal genewere analyzed further.

From strain ATX16538, pAX2579 was found to contain an open reading framewith homology to “cry” type delta-endotoxins. This open reading framewas designated as axmi-039 (SEQ ID NO:3), and the encoded protein wasdesignated AXMI-039 (SEQ ID NO:4). The axmi-039 ORF and flankingsequence (151 bp upstream of the start codon and 29 bp downstream of thestop codon) was PCR amplified, cloned into pRSF1B and sequenced to yieldpAX2579. pAX2579 was deposited with the ARS Patent Strain Collection onJun. 9, 2006, and assigned NRRL B-30936. AXMI-039 is 43.9% amino acidsequence identity to Cry5Ba1, which is the closest homolog identified.

From strain ATX16093, pAX4313 was found to contain an open reading framewith homology to “cry” type delta-endotoxins. This open reading framewas designated as axmi-040 (SEQ ID NO:5), and the encoded protein wasdesignated AXMI-040 (SEQ ID NO:6). pAX4313 was deposited with the ARSPatent Strain Collection on Jun. 9, 2006, and assigned NRRL B-30937.AXMI-040 is 42.9% amino acid sequence identity to Cry21Ba1, which is theclosest homolog identified.

From strain ATX21049, an open reading frame was identified thatexhibited homology to “cry” type delta-endotoxins. This open readingframe was designated as axmi-049 (SEQ ID NO:34), and the encoded proteinwas designated AXMI-049 (SEQ ID NO:35). This open reading frame wasamplified by PCR and cloned into a vector to yield pAX5039. pAX5039 wasdeposited with the ARS Patent Strain Collection on May 29, 2007, andassigned NRRL B-50046. AXMI-049 has 46.1% amino acid sequence identityto Cry21Ba1, which is the closest homology identified.

Example 19 Vectoring of the Pesticidal Genes of the Invention for PlantExpression

Each of the coding regions of the genes of the invention are connectedindependently with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinII terminators.Techniques for producing and confirming promoter—gene—terminatorconstructs also are well known in the art.

Example 20 Transformation of the Genes of the Invention into Plant Cellsby Agrobacterium-Mediated Transformation

Ears are collected 8-12 days after pollination. Embryos are isolatedfrom the ears, and those embryos 0.8-1.5 mm in size are used fortransformation. Embryos are plated scutellum side-up on a suitableincubation media, and incubated overnight at 25° C. in the dark.However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors for Ti plasmid mediated transfer for 5-10 min, andthen plated onto co-cultivation media for 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor five days (at 25° C. in the dark). Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

Example 21 Transgenic Plants Expressing AXMI-031 and Variants

The plant expression cassettes described herein are combined with anappropriate plant selectable marker to aid in the selections oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation. synaxmi-031(apo) was cloned into a plant expressionvector, and this vector was introduced into Agrobacterium tumefaciens asknown in the art. The synaxmi-031(apo) coding region under control ofthe Arabidopsis UBQ3 promoter (Norris et al. (1993) J. Plant Mol. Biol.21:895-906) was introduced into Arabidopsis thaliana by floral dipmethod as known in the art, and transgenic plants were selected. Thepresence of synaxmi-031(apo) in the transgenic T1 population wasconfirmed by PCR analysis as known in the art, using oligonucleotideprimers derived from the sequence of synaxmi-031, which also anneal tosynaxmi-031(apo).

Leaf and root tissue from transgenic synaxmi-031(apo) containing plantswas prepared and separated on polyacrylamide gels alongsidenon-transgenic controls and dilutions of AXMI-031 protein, andtransferred to a membrane as known in the art. The samples were testedfor the presence of the expression product of synaxmi-031(apo) byWestern blot analysis as known in the art, using anti-AXMI-031antibodies described herein. The expression product of synaxmi-031(apo)of the expected size was detected in samples of both leaf and roottissue from transgenic synaxmi-031(apo)-containing plants, but notdetected in non-transgenic controls.

TABLE 5 Antibody detection of SYNAXMI- 031(APO) in transgenic plantsDetection of SYNAXMI- Source of Tissue 031(APO) Non-transformed control− synaxmi-031(apo) +++ containing plant ‘A’ synaxmi-031(apo) +++containing plant ‘B’

Example 22 Reduced Cyst Formation by AXMI-031 Expressing Plants

Transgenic plants expressing synaxmi-031(apo) were tested for ability toresist infestation by Heterodera schachtii compared to control plants.Transgenic plants, as well as control plants transformed with vectoralone, were infested with approximately 100 J2 hatchlings, and thenumber of nematodes entering the roots, as well as the number of cystformed, was measured. Transgenic plants expressing SYNAXMI-031(APO) werefound to consistently have reduced numbers of nematodes entering theroots, and reduced numbers of cysts formed relative to controls.

TABLE 6 Reduced cyst formation in AXMI-031 expressing plants Cystformation Control plant ++ Plants expressing AXMI-031(APO) +

Example 23 Additional Assays for Pesticidal Activity

The ability of a pesticidal protein to act as a pesticide upon a pest isoften assessed in a number of ways. One way well known in the art is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested, or controlsamples. Often this is performed by placing the material to be tested,or a suitable dilution of such material, onto a material that the pestwill ingest, such as an artificial diet. The material to be tested maybe composed of a liquid, solid, or slurry. The material to be tested maybe placed upon the surface and then allowed to dry. Alternatively, thematerial to be tested may be mixed with a molten artificial diet; then,dispensed into the assay chamber. The assay chamber may be, for example,a cup, a dish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson, J. L. & H. K. Preisler. 1992.Pesticide bioassays with arthropods. CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals “ArthropodManagement Tests” and “Journal of Economic Entomology” or by discussionwith members of the Entomological Society of America (ESA).

Example 24 Transformation of Maize Cells with the Pesticidal Genes ofthe Invention

Maize ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol;1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express the genes of the invention in plantcells are accelerated into plant tissue using an aerosol beamaccelerator, using conditions essentially as described in PCTPublication No. WO/0138514. After beaming, embryos are incubated for 30min on osmotic media, then placed onto incubation media overnight at 25°C. in the dark. To avoid unduly damaging beamed explants, they areincubated for at least 24 hours prior to transfer to recovery media.Embryos are then spread onto recovery period media, for 5 days, 25° C.in the dark, then transferred to a selection media. Explants areincubated in selection media for up to eight weeks, depending on thenature and characteristics of the particular selection utilized. Afterthe selection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

Materials

DN62A5S Media Components per liter Source Chu'S N6 Basal 3.98 g/LPhytotechnology Labs Salt Mixture (Prod. No. C 416) Chu'S N6 Vitamin 1mL/L Phytotechnology Labs Solution (Prod. (of 1000× Stock) No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/LSigma D-7299) (of 1 mg/mL Stock)

Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KCl, addGelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C., add 2ml/L of a 5 mg/ml stock solution of Silver Nitrate (PhytotechnologyLabs). Recipe yields about 20 plates.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A recombinant nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) the nucleotide sequence of SEQID NO:34; b) a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:35; c) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO:35,wherein said amino acid sequence has pesticidal activity against anematode pest; and, d) the delta endotoxin nucleotide sequence of theDNA insert of the plasmid deposited at the Northern Regional ResearchLaboratory (NRRL) as Accession No. B-50046.
 2. The recombinant nucleicacid molecule of claim 1, wherein said nucleotide sequence is asynthetic sequence that has been designed for expression in a plant. 3.A vector comprising the nucleic acid molecule of claim
 1. 4. The vectorof claim 3, further comprising a nucleic acid molecule encoding aheterologous polypeptide.
 5. A host cell that contains the recombinantnucleic acid molecule of claim
 1. 6. The host cell of claim 5 that is abacterial host cell.
 7. The host cell of claim 5 that is a plant cell.8. A transgenic plant comprising the host cell of claim
 7. 9. Thetransgenic plant of claim 8, wherein said plant is selected from thegroup consisting of maize, sorghum, wheat, cabbage, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,tobacco, barley, and oilseed rape.
 10. A transgenic seed comprising thenucleic acid molecule of claim
 1. 11. A plant having stably incorporatedinto its genome a DNA construct comprising a nucleotide sequence thatencodes a protein having pesticidal activity, wherein said nucleotidesequence is selected from the group consisting of: a) the nucleotidesequence of SEQ ID NO:34; b) a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:35; c) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:35, wherein said amino acid sequence haspesticidal activity against a nematode pest; and, d) the delta endotoxinnucleotide sequence of the DNA insert of the plasmid deposited at theNRRL as Accession Nos. B-50046; wherein said nucleotide sequence isoperably linked to a promoter that drives expression of a codingsequence in a plant cell.
 12. The plant of claim 11, wherein said plantis a plant cell.
 13. A method for protecting a plant from a pest, saidmethod comprising introducing into said plant or cell thereof at leastone expression vector comprising a nucleotide sequence that encodes apesticidal polypeptide, wherein said nucleotide sequence is selectedfrom the group consisting of: a) the nucleotide sequence of SEQ IDNO:34; b) a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:35; c) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO:35,wherein said amino acid sequence has pesticidal activity against anematode pest; and, d) the delta endotoxin nucleotide sequence of theDNA insert of the plasmid deposited at the NRRL as Accession Nos.B-50046.
 14. The recombinant nucleic acid sequence of claim 1, whereinsaid nucleic acid sequence is operably linked to a promoter that drivesexpression of said nucleic acid sequence in a plant cell.